Throughout this application various publications are referenced by numbers within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications, in their entireties, are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
Corneal endothelial fluid transport: This invention originates from prior studies on water relations in the cornea of the eye. This organ is kept transparent by the activity of its posterior epithelial monocellular cell layer, the endothelium, which displays a high osmotic permeability [1,2] and transports significant amounts of electrolytes and water [3,4] across itself. In the course of the studies disclosed herein on the basic mechanism of this fluid transport [4], recent evidence has [2] led us to conclude that the transendothelial transport of water most probably takes place across the cell membranes, just as it appears to do in other water-transporting epithelia such as the gall bladder [5].
The route for water permeation through cell membranes: In order to explain fluid transport fully, one would need to know the basic mechanism of water movements through cells and tissues. Yet, although water movements across cell membranes are a most ubiquitous and important feature of living processes, the precise route for it remains unclear. Thus, it has been variously suggested (6) that water simply traverses the lipid bilayer in most membranes except for those most permeable, where it would in addition traverse a water pore (7), also called a water channel (8), which would constitute a polar route across the lipid bilayer.
Studies of this latter route in human erythrocyte membranes had led to the suggestion (9,10) that the transmembrane protein (band 3) containing the anion channel (11, 12) is the route of the water transport. However, later evidence has cast doubts on such suggestion. For example, 5,5-dithiobis (2-nitrobenzoate), (DTNB), which is a good blocker of and marker for the anion channel in red blood cells, has been variously reported to block osmotic water flow by 60% (13) and not to affect water permeability (14). This discrepancy is important, since the observed inhibitory effect of DTNB on osmotic flow was central for the argument that the anion channel is the water permeation route (10). More recent evidence has pointed to either the anion channel or the glucose transporter or both (bands 3.0 and 4.5) as the water permeation site. (15, 16); still more definite identification could not be done for lack of evidence that any specific inhibitor of those proteins would block water transport.
The glucose transporter as water channel: Given this background, reexamination has been made of the effect on osmotic water flow of blockers of sulfhydryl groups and of both anion and glucose permeation. Two of the approaches utilized appear to be novel for this area of work:
(a) water flow was measured across an epithelial preparation (the corneal endothelium) instead of red blood cells, and, PA1 (b) rather than determining osmotically-induced cell volume changes (as done classically with red blood cells), the rate of water flow as a function of time was monitored continuously.
While some prior results obtained with erythrocytes were confirmed, a crucial difference also was found, namely, that in the preparation all glucose transport inhibitors blocked osmotic water flow. The conclusion that emerged from these experiments is consistent with the hypothesis that both water and glucose traverse cell membranes through the same channel-like pathway, that is, through the protein identified as the glucose transporter (band 4.5). An account of some of these early findings has appeared in Abstract form [17].